Mycophenolic acid (MPA) was initially isolated from a culture of Penicillium (B. Gosio, Riv. Igiene Sanita Pub. Ann, 7, 825–869, 1896). Its value as an immunomodulator was realized much later. Morpholino ester of mycophenolic acid is used as a prodrug in pharmaceutical composition for treatment of rheumatoid arthritis, psoriasis and in prevention of tissue rejection in organ transplant patients.
Mycophenolic acid is produced by aerobic fermentation of several Penicillium species. It has a broad spectrum of activity like antitumor activity, antiviral, antipsoriatic, immunosuppressive and anti-inflammatory activity. It also exhibits antibacterial and antifungal activities. It is tolerable in large doses and has minimal side effects. It inhibits inosine monophosphate dehydrogenase which is an important enzyme in de novo synthesis of inosine monophosphate, a precursor of purines. MPA also inhibits proliferation of lymphocytes that are responsible for immune response. This immuno repressory effect of mycophenolic acid has been important in treatment of organ rejection after organ transplant surgery.
There is a continuous need to find improved process for production of mycophenolic acid that will be commercially viable. It has been shown that mutants Penicillium brevi-compactum resistant to polyene antibiotics, HMG CoA reductase inhibitors, methyl viologen and surfactants produce more MPA than the parent strain (U.S. Pat. No. 4,452,891).
The proposed biosynthetic pathway of mycophenolic acid production involves steroid biosynthetic pathway as well as polyketide biosynthetic pathway. It is a condensation product of a tetraketide and geranyl pyrophosphate, wherein geranyl moiety is cleaved after condensation and subsequent o-methylation in the tetraketide ring yields mycophenolic acid (Muth and Nash, Antimicrobial Agents and Chemotherapy, 8, 321–327, 1896). The biosynthetic pathway was studied in P. stoloniferum. 
Penicillium brevi-compactum strain has been used in submerged fermentation where it produces 2.4 mg/ml at 27° C. in 6 days on shaking and gives 3.6 mg/ml at 27° C. in 14 days without shaking (U.S. Pat. No. 4,452,891). In solid substrate fermentation (SSF) it is reported to produce 3286 mg per Kg of wheat bran (Sadhukhan et al, J. Ind. Microbiol. Biotechnol. 22, 33–38, (1999). Regarding the economics of using a producing strain, the described volume-time-yields as an industrial process are not economically attractive.
The object of the present invention is to provide an improved method for the production of mycophenolic acid by solid substrate fermentation in a novel bioreactor ‘PLAFRACTOR’ and its subsequent purification.
To achieve the said objective this invention provides an improved method for the manufacture of Mycophenolic acid comprising:                loading a contained bioreactor with solid substrate matrix and sterilizing it,        mixing the said sterilized solid substrate matrix with Penicillium brevi-compactum,         adding 5–20% of glycerol, if desired,        incubating the said inoculated solid substrate matrix for 4–7 days at 20–35° C.,        extracting the fermented solid substrate matrix with an organic solvent,        concentrating the organic solvent extract,        crystallizing mycophenolic acid by adjusting the pH to about 2.0 with an inorganic acid and allowing to stand for about 3 hours, followed by filtration using a filter aid,        dissolving the filter aid cake in a water immiscible organic solvent and treating with alumina,        filtering the water immiscible organic solvent followed by concentration of the organic layer by distillation,        dissolving the concentrate obtained in an alcohol,        dispersing the alcoholic solution in water and filtering to get the crude crystals,        dissolving the crude crystals thus obtained in an organic solvent,        adding another organic solvent to the previous step solution and chilling to 4 to −20° C. to get pure mycophenolic acid crystals.        
The Penicillium brevi-compactum used is in the form of spore suspension or in mycelial form.
The solid substrate matrix is selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk.
The solid substrate matrix is a mixture of two or more solid substances selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk.
The said contained bioreactor allows solid state fermentation to be carried out in a manner such that the fermentation micro-organisms and the fermentation products produced are kept isolated from the outside environment during the course of fermentation. The said contained bioreactor is “PLAFRACTOR”.
The organic solvent used for extraction is selected from acetone, ethanol, toluene, benzene or ethyl acetate.
The filter aid is selected from celite, perlite or alumina.
The solvent used to dissolve the filter aid cake is selected from cyclohexane, toluene, benzene, ethyl acetate or butyl acetate.
The alcohol used for crystallization is selected from methanol, ethanol or iso-propanol.
The inorganic acid used for adjusting pH is sulphuric acid and the concentration of the organic layer is carried out by azeotropic distillation.
The present invention uses Penicillium brevi-compactum. The colony of this isolate was comparatively fast growing and the aerial mycelium was cottony and white. The microbial culture sporulated showing green coloration on third day.
The bioreactor used for the solid substrate fermentation is our invention and is described in our PCT publication no. WO 00/29544. The said bioreactor is modular in nature and carries out all of the processes of solid substrate fermentation in a single contained environment. The modular construction of the bioreactor provides multiple modules stacked on top of one another, each with a base connected to frame for holding the solid medium in isolation from the exterior environment. The construction of the bioreactor allows solid substrate fermentation to be carried out in a manner such that the fermenting microorganisms and the fermentation products it produces are kept isolated from the outside environment during the course of the fermentation. This containment of the fermentation process is of significant importance when working with microbial metabolites, which are cytotoxic in nature e.g. Cyclosporin, mycophenolic acid.
The said bioreactor operates in a contained manner and is capable of sterilizing the solid state fermentation media, cooling it to the required temperature, fermenting at the desired set conditions, in situ extraction of the end product, recovery of the solvents and post harvest sterilization.
An important aspect of the bioreactor is a mechanism of heat removal resulting in stringent temperature control of the fermentation process. In comparison, maintaining a constant temperature of growth in solid substrate fermentation using tray cultures is not efficient. The base plate of the bioreactor has multiple channels called noncommunicating channels that carry heating and cooling fluids sandwiched between two sheets. Heat is transferred to and from the modules by conduction. In this way the temperature of the module is precisely maintained to meet the specific requirement of different microorganisms.
The base of the module contains a second set of channels, the communicating channels to deliver sterile air as supply of oxygen into the solid substrate bed for optimum growth of organism. Moisture loss because of passage of sterile air is significantly reduced by regularly reversing the direction of airflow every few hours. Using this, homogeneity in moisture content is maintained throughout the bioreactor. These aspects provide ample convenience over previous SSF methodologies that require multiple manipulations at each step of the fermentation process.
The invention will now be described with reference to the following examples: